Sodium reabsorbing epithelia, such as renal, distal, and collecting tubules, have as their major function the control of whole-body sodium balance. These epithelia contain apical membrane Na + channels that are inhibited by the diuretic amiloride. It is at the level of these channels that the feedback control mechanisms necessary for the maintenance of Na + homeostasis occur. The long-term goal of this project remains to elucidate at the molecular level the mechanisms responsible for the regulation of ion flow through these conductive entry pathways. During the previous grant period four novel observations were made that form the basis of this continuation application. First, we discovered that Ca 2v was involved in the effect on conductance following the interaction of actin with ENaC. Second, a short, 14-aa segment in the C-terminal of alpha-ENaC was identified as being crucial for actin's functional interaction with ENaC. Third, we have identified new functional and physical interactions between ENaC, syntaxin, and other novel cytoplasmic regulatory elements. Fourth, we have utilized the baculovirus system to produce milligram quantities of pure, functionally intact alpha-ENaC. Therefore, we propose to 1) test the hypothesis that t-SNARES (e.g., syntaxin 1A) and annexins directly modulate ENaC function; and 2) test the hypothesis that actin directly binds to ENaC, thereby inducing a conformational change resulting in changes in channel conductance and cation selectivity. We will identify the site of physical contact between ENaC and syntaxin, actin, annexins, and other cytoskeletal linking elements such as ezrin. Proteins that regulate the activity of syntaxin, such as SNAP 23/25 and munc-18, will also be examined for their functional and physical influences on syntaxin-ENaC interactions. We will also crystallize a-ENaC with the goal of providing a detailed molecular picture of this subunit. These results will offer new insights into the nature and regulation of amiloride-sensitive Na+ channels, the ways that these channels interact with and are modulated by the cytoskeleton, and provide the first near atomic-level detail of these important ion channels. Thus, understanding the molecular basis for ENaC regulation, conduction, and selectivity will provide unique opportunities for therapeutic interventions in an ever-increasing plethora of ENaC-related diseases.